Throughout this application, various publications are referenced by Arabic numerals within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
The life cycle of animal viruses is characterized by a series of events that are required for the productive infection of the host cell. The initial step in the replicative cycle is the attachment of the virus to the cell surface which is mediated by the specific interaction of the viral attachment protein (VAP) to receptors on the surface of the target cell. The pattern of expression of these receptors is largely responsible for the host range and tropic properties of viruses. The interaction of the VAP with cellular receptors therefore plays a critical role in infection and pathogenesis of viral diseases and represents an important area to target the development of anti-viral therapeutics.
Cellular receptors may be comprised of all the components of membranes, including proteins, carbohydrates, and lipids. Identification of the molecules mediating the attachment of viruses to the target cell surface has been made in a few instances. The most extensively characterized viral receptor protein is CD4 (T4) (1). CD4 is a nonpolymorphic cell surface glycoprotein that is expressed primarily on the surface of helper T lymphocytes and cells of the monocyte/macrophage lineage. CD4 associates with major histocompatibility complex (MHC) class II molecules on the surface of antigen-presenting cells to mediate efficient cellular immune response interactions. In man, CD4 is also the target of interaction with the human immunodeficiency virus (HIV).
HIV infects primarily helper T lymphocytes and monocytes/macrophages, cells that express surface CD4, leading to a gradual loss of immune function which results in the development of the human acquired immune deficiency syndrome (AIDS). The initial phase of the HIV replicative cycle involves the high affinity interaction between the HIV exterior envelope glycoprotein gp120 and surface CD4 (Kd approximately 4.times.10.sup.-9 M) 2). Several lines of evidence demonstrate the requirement of this interaction for viral infectivity. In vitro, the introduction of a functional cDNA encoding CD4 into human cells which do not express CD4 is sufficient to render otherwise resistant cells susceptible to HIV infection (3). In vivo, viral infection appears to be restricted to cells expressing CD4. Following the binding of HIV gp120 to cell surface CD4, viral and target cell membranes fuse, resulting in the introduction of the viral capsid into the target cell cytoplasm.
Characterization of the interaction between HIV gp120 and CD4 has been facilitated by the isolation of cDNA clones encoding both molecules (4, 5). CD4 is a nonpolymorphic, lineage-restricted cell surface glycoprotein that is a member of the immunoglobulin gene superfamily. High-level expression of both full-length CD4 and truncated, soluble versions of CD4 (sCD4) have been described in stable expression systems. The availability of large quantities of purified sCD4 has permitted a detailed understanding of the structure of this complex glycoprotein. Mature CD4 has a relative molecular mass (Mr) of 55 kilodaltons and consists of an amino-terminal 372 amino acid extracellular domain containing four tandem immunoglobulin-like regions denoted V1-V4, followed by a 23 amino acid transmembrane domain and a 38 amino acid cytoplasmic segment. The amino-terminal immunoglobulin-like domain V1 bears 32% homology with kappa light chain variable domains. Three of the four immunoglobulin-like domains contain a disulphide bond (V1, V2 and V4), and both N-linked glycosylation sites in the carboxy-terminal portion of the molecule are utilized (4, 6).
Experiments using truncated sCD proteins demonstrate that the determinants of high-affinity binding to HIV gp120 lie within the amino-terminal immunoglobulin-like domain V1 (7-9). Mutational analysis of V1 has defined a discrete gp120 binding site (residues 38-52 of the mature CD4 protein) that comprises a region structurally homologous to the second complementarity-determining region (CDR2) of immunoglobulins (9). The production of large quantities of V1V2 has permitted a structural analysis of the two amino-terminal immunoglobulin-like domains. The structure determined at 2.3 angstrom resolution reveals that the molecule has two tightly associated domains containing the immunoglobulin-fold connected by a continuous beta strand. The putative binding sites for monoclonal antibodies, class II MHC molecules and HIV gp120 (as determined by mutational analysis) map on the molecular surface (10, 11).
A soluble version of the entire extracellular segment of CD4 (V1-V4, termed sCD4) has been described and appears to be a potential therapeutic approach to the treatment of HIV infection 12). In vitro experiments demonstrate that: 1) SCD4 acts as a "molecular decoy" by binding to HIV gp120 and inhibiting viral attachment to and subsequent infection of human cells; 2) sCD4 "strips" the viral envelope glycoprotein gp120 from the viral surface; and 3) sCD4 blocks the intercellular spread of virus from HIV-infected cells to uninfected cells by inhibiting virus-mediated cell fusion (1, 13).
In addition to in vitro results, experiments with sCD4 in simian immunodeficiency virus (SIV)-infected rhesus monkeys have been described. These studies demonstrated that administration of 2 milligrams (intramuscular) of sCD4 for 28 days to SIV-infected rhesus monkeys led to a decreased ability to isolate virus from peripheral blood lymphocytes and bone marrow. In addition, the growth of granulocyte-macrophage and erythrocyte progenitor colonies in the bone marrow returned to normal levels. These data suggest that administration of sCD4 to SIV-infected rhesus monkeys leads to a diminution of the viral reservoir.
Phase I human clinical trials demonstrated that there is no significant toxicity or immunogenicity associated with administration of sCD4 at does as high as 30 mg/day. Pharmocokinetic studies revealed the serum half-life of sCD4 to be 45 minutes following intravenous administration, 9.4 hours after intramuscular dosing, and 10.3 hours after the drug was given subcutaneously (14, 15). Preliminary antiviral studies were inconclusive with respect to CD4 cell count and levels of HIV antigen. Because the maximum tolerated dose was not reached, the antiviral effect of sCD4 may have been underestimated, especially in light of recent data concerning differences in sCD4 concentrations required to inhibit laboratory strains of HIV-1 compared to primary viral isolates (16).
Although these in vitro, primate, and human clinical studies with sCD4 have produced encouraging results, they have also defined several limitations. First, the measured serum half-life of sCD4 is relatively short. Second, sCD4 is monovalent with respect to gp120 binding in contrast with cell surface CD4 and viral surface gp120 which are multivalent. Third, sCD4 is not cytotoxic for HIV-infected cells. Fourth, sCD4 may not cross the placenta to a significant degree. Therefore, chimeric CD4 molecules have been described which take advantage of the immunoglobulin-like nature of CD4 and several beneficial properties of immunoglobulins themselves (i.e. CD4-immunoglobulin fusions).
Immunoglobulins, or antibodies, are the antigen-binding molecules produced by B lymphocytes which comprise the humoral immune response. The basic unit of an immunoglobulin molecule consists of two identical heavy chains and two identical light chains. The amino-terminus of each chain contains a region of variable amino acid sequence (variable region). The variable regions of the heavy and light chains interact to form two antigen binding sites. The carboxy-terminus of each chain contains a region of constant amino acid sequence (constant region). The light chain contains a single constant domain, whereas the heavy chain constant domain is subdivided into four separate domains (CH1, hinge, CH2, and CH3). The heavy chains of immunoglobulin molecules are of several types, including mu (M), delta (D), gamma (G), alpha (A) and epsilon (E). The light chains of immunoglobulin molecules are of two types, either kappa or lambda. Within the individual types of heavy and light chains exist subtypes which may differ in effector function. An assembled immunoglobulin molecule derives its name from the type of heavy chain that it possesses.
The development of monoclonal antibodies has circumvented the inherent heterogeneity of antibodies obtained from serum of animals or humans. However, most monoclonal antibodies are derived from cells or mouse origin and therefore are immunogenic when administered to humans. More recent developments combining the techniques of molecular genetics with monoclonal antibody technology has lead to the production of "humanized" chimeric antibodies in vitro. In these chimeric antibodies, the variable domains of human immunoglobulin heavy and light chains are replaced with specific heavy and light chain variable domains from a murine monoclonal antibody (17-19). The result of this genetic manipulation is a molecule with specificity for a particular antigen and the characteristics of human immunoglobulins.
Sequence and structural analyses of CD4 indicate that the four extracellular domains are immunoglobulin-like. Since the Fc portion of immunoglobulins controls the rate of catabolism of the molecules (serum half-life ranging from 14 to 21 days) and provides various effector functions, several reports describe the replacement of variable and constant domains of immunoglobulins with the immunoglobulin-like domains of CD4 (21-24).
CD4-IgG1 heavy chain fusion proteins resulting in chimeric gamma1 heavy chain dimers have been described (21). These molecules contain the gamma1 heavy chain CH1 domain in addition to the hinge, CH2 and CH3 domains. However, heavy chain assembly and secretion from mammalian cells is less efficient if the CH1 domain is expressed in the absence of light chains (25). Subsequently, a CD4-IgG1 heavy chain fusion protein lacking the CH1 domain and the first five amino acids of the hinge region was described which was secreted to high levels (22). These fusion proteins retain various effector functions of immunoglobulin molecules, such as Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC) toward HIV-1-infected cells, and placental transfer via an Fc receptor-dependent mechanism (22). CD4-IgM heavy chain fusion proteins have also been described (26). In addition, CD4-IgG1 fusion proteins have been described wherein the V1V2 domains of CD4 are fused to the CH1, hinge, CH2 and CH3 domains of a gamma1 heavy chain, and wherein the V1V2 domains of CD4 are fused to the constant domain of a kappa light chain (29).
Fusion proteins linking CD4 to toxins have also been constructed and tested for their ability to kill HIV-infected cells. In one study, sCD4 was coupled to the deglycosylated A chain of ricin which inactivities ribosomes, therefore inhibiting protein synthesis and killing the cell (27). This fusion protein was reported to specifically lyse cells infected with five different isolates of HIV, but was nontoxic to uninfected cells. In another study, the V1V2 domains of CD4 were coupled to domains II and III of Pseudomonas exotoxin A (28). This fusion protein was reported to specifically bind and inhibit protein synthesis in cells expressing the HIV envelope glycoprotein gp120 (25).
It is well established that human monocytes and macrophages (M/M) express surface CD4, can be infected by HIV, and serve as a reservoir of infection and a vehicle for viral dissemination (29). Furthermore human M/M also contain Fc receptors, which are responsible for binding to specific IgG molecules via their Fc portion (see Table 1). The high affinity Fc receptor (FcRI) binds monomeric IgG and complexed IgG (antigen plus antibody). The rank order of affinity of FcRI for IgG isotypes is IgG1=IgG3&gt;IgG4, and does not interact with IgG2. The low affinity Fc receptor (FcRII) binds monomeric IgG with lower affinity than IgG in complexed form. The rank order of affinity is that IgG1 and IgG3 binding is greater than that of Ig2 or IgG4 (30).
Affinity Molecular for FcReceptor Weight Affinity Expression isotypes FcRI 72,000 High Monocytes IgG1, IgG3&gt; IgG4, does not bind IgG2 FcRII 40,000 Low Monocytes, IgG1, IgG3&gt; platelets, IgG2, IgG4 neutrophils FcRIII 50-70,000 Low Neutrophils IgG1, IgG3 NK, K, monocytes
(Table abbreviated from Gergely J. and Sarmay G. (1990) FASEB J. 4:3275
Because of the recent demonstration that HIV+ patients' sera contain low titer antibodies which recognize the HIV envelope glycoprotein, it has been observed that infection of M/M is enhanced by low titer anti-HIV antibodies, presumably by cross bridging HIV and the Fc receptor (31). Enhance infection of macrophages by Dengue virus, Yellow fever virus, and Sindbis virus, is well documented in vitro as well as in Rhesus monkeys (32). Such enhancements has been demonstrated to occur in the presence of subneutralizing antibodies to these viruses, which serves to opsonize the viruses and bind them to the FcRs (or complement receptors) on the surface of the cell. In the case of HIV, this crossbridging serves to concentrate HIV onto the surface of the M/M, whereupon the virus is then able to utilize CD4 for entry into the cell, since sCD4 is able to inhibit the enhancement seen with low titer antibodies (31).
Recently, Byrn et al. (22) have produced a CD4-IgG chimera of the IgG1 isotype, to increase the plasma half-life of sCD4 as well as to confer effector functions to the chimeric molecule. Therefore this molecule has the potential to bind to Fc receptors located on the surface of the M/M, and potentially cause an increase in the infection of these cell types. Because enhanced infection of these cell types is a serious consideration in developing novel therapeutics, our objective for designing a CD4-IgG molecule was to use the IgG2 type, which has a greatly diminished ability to bind M/M Fc receptors (30). Furthermore, human IgG2 antibodies appear to lack significant allotypic variation, whereas human IgG1 antibodies contain allotypic variations (33). Therefore, to avoid potential immunogenic responses to recombinant molecules containing immunoglobulin domains, we have chosen a molecule which is the least polymorphic and has a decreased ability to concentrate HIV onto the surface of the macrophage.
Second, similar observations of enhanced infection of unborn babies may also be demonstrated for CD4-IgG1 immunoadhesions administered to pregnant mothers. For example, it is well documented that the placental syncytiotrophoblast plasma membrane contains Fc receptors (30). Because materno-fetal transport of immunoglobulin is primarily restricted to the IgG class, it is believed that passive immunity can be achieved by specific transport across the placenta via a specific Fc receptor transcytotic mechanism. Further, it appears that the Fc receptors on the placental syncytiotrophoblast membrane are selective in that immunoglobulins of the IgG1 type have approximately 10-20 fold higher binding affinity for the receptor. In fact, of all the IgG subtypes, IgG1 and 3 have the highest affinity for the receptor, followed by IgG4, and finally IgG2 (30). These results are consistent with those obtained from the cloning of the FcR from a human placenta, which indicate that the receptor is very similar to the FcRII type found on M/M. Although one might argue that transplacental transport of immunoglobulin may be beneficial to the fetus in utero, it could also be argued that specific maternal immunoglobulin raised to a specific pathogen (such as HIV), might facilitate transport across the placenta via an Fc dependent mechanism, to increase infection of the fetus, similar to the mechanism which has evolved to transport IgA across epithelia, via the poly Ig receptor (34). Thus specific CD4-IgG1 fusion proteins, which have been demonstrated to cross the placenta and concentrate in the fetal blood (22), may be detrimental to the fetus, by providing HIV with a novel mechanism to cross the placental barrier.
We have now discovered that a specific CD4-gamma2 chimeric heavy chain homodimer provides advantages relative to those CD4-IgG1 heavy chain homodimers which have been described more than one year ago. Specifically, we have constructed a CD4-gamma2 chimeric heavy chain homodimer which contains the V1V2 domains of CD4 and which is efficiently assembled intracellularly and efficiently secreted from mammalian cells as a homodimer, enabling high recovery and purification from the medium of cells expressing this chimeric heavy chain homodimer. To construct this homodimer, we have used the entire hinge, CH2, and CH3 domains from a human gamma2 heavy chain, which results in a chimeric molecule containing the constant domains of a human IgG2 molecule responsible for dimerization and efficient secretion. This is in contrast to the heavy chain dimers described by Capon and Gregory (20) which include the CH1 domain in the CD4-IgG1 heavy chain dimer, resulting in poor secretion and recovery from cell culture medium of the recombinant molecule. We have also included the entire hinge domain of gamma2 heavy chain in the CD4-gamma2 chimeric heavy chain homodimer of this invention to provide efficient dimerization, since the cysteine residues contained in this domain are responsible for forming the disulphide links to the second chain of the homodimer, positioning the two chains in the correct spatial alignment and facilitating formation of the antigen combining site.
Furthermore, by including the entire hinge domain, we have maintained the segmental flexibility of the heavy chain dimers, thus enabling modulation of biological function such as complement activation and Fc receptor binding (29).
Since IgG2 immunoglobulins have a greatly diminished ability to bind to Fc receptors on monocytes, macrophages, and placental membranes, construction of a CD4-gamma2 chimeric heavy chain homodimer and a CD4-IgG2 chimeric heterotetramer results in chimeric proteins with many advantages that CD4-gamma1 chimeric heavy chain homodimers or CD4-IgG1 chimeric heterotetramers may not possess (20, 23, 24, 26). Furthermore, human IgG2 is significantly less polymorphic than other IgG types and therefore is less likely to be immunogenic when administered to humans. This is in contrast to human IgG1 which contain many allotypes and has a higher probability of being immunogenic when administered to humans.
In addition to the CD4-gamma2 chimeric heavy chain homodimers, we have also constructed CD4-IgG2 heavy chains, which contain the V1V2 domains of CD4 fused to the CH1, hinge, CH2 and CH3 domains of human gamma2 heavy chain. These molecules encode a CD4-IgG2 chimeric heterotetramer, and when co-expressed in the presence of CD4-kappa chimeric light chains containing the V1 and V2 domains of CD4 fused to the entire constant domain of human kappa light chains (or lambda light chains), enable the production of said heterotetramer. This heterotetramer comprises two CD4-IgG2 chimeric heavy chains and two CD4-kappa chimeric light chains. Producing heavy chains which contain the CH1 domain enables efficient association with the CD4-kappa chimeric light chains, resulting in efficient secretion of a CD4-IgG2 chimeric heterotetramer. These CD4-IgG2 chimeric heterotetramers possess increased serum half-lives and increased avidity for HIV as compared with heavy chain dimers.